An antioxidation strategy based on ultra-small nanobubbles without exogenous antioxidants

Antioxidation is in demand in living systems, as the excessive reactive oxygen species (ROS) in organisms lead to a variety of diseases. The conventional antioxidation strategies are mostly based on the introduction of exogenous antioxidants. However, antioxidants usually have shortcomings of poor stability, non-sustainability, and potential toxicity. Here, we proposed a novel antioxidation strategy based on ultra-small nanobubbles (NBs), in which the gas–liquid interface was employed to enrich and scavenge ROS. It was found that the ultra-small NBs (~ 10 nm) exhibited a strong inhibition on oxidization of extensive substrates by hydroxyl radicals, while the normal NBs (~ 100 nm) worked only for some substrates. Since the gas–water interface of the ultra-small NBs is non-expendable, its antioxidation would be sustainable and its effect be cumulative, which is different to that using reactive nanobubbles to eliminate free radicals as the gases are consumptive and the reaction is unsustainable. Therefore, our antioxidation strategy based on ultra-small NB would provide a new solution for antioxidation in bioscience as well as other fields such as materials, chemical industry, food industry, etc.

www.nature.com/scientificreports/ study the antioxidation or oxidation of a gas-liquid interface. Previously, it has been reported that oxygen NBs promoted the formation of ROS by producing hydroxyl radicals through the collapse of the microbubbles 34 , while the reductive hydrogen NBs helped the quenching of ROS 35,36 . However, in these studies, the chemical properties of the gas phases rather than the size of NBs were focused on, in which the gases in the nanobubbles are consumptive and would run out so that the redox reaction is unsustainable. In this study, an antioxidation strategy based on ultra-small NBs without exogenous antioxidants was provided. A significant size dependence was observed when the NBs were employed to determine their ability to block the oxidization of substances by the hydroxyl radicals. It was found that the ultra-small NBs exhibited a strong antioxidant effect for extensive substrates, while normal NBs worked only for some substrates. Since the gas-water interface of the ultra-small NBs is non-expendable, its antioxidation would be sustainable and its effect be cumulative. We believe that this research would help develop new solutions for removing excess free radicals in a system without reductants supply.

Results
Antioxidation of ultra-small N 2 NBs. The experiment was first conducted by determining the antioxidant effect of the ultra-small nitrogen (N 2 ) NBs by detecting their ability to block the oxidization of 3, 3′, 5, 5′-tetramethylbenzidine (TMB) caused by the hydroxyl radicals (Figures S1 and S2) generated from H 2 O 2 with the catalysis of Cu 2+ . Ultra-small N 2 NBs were generated in cold pure water (0 °C) during a compressiondecompression process 37 and then were introduced to the oxidation-reaction system under room temperature and atmospheric pressure. The oxidation curves were obtained by monitoring the absorbance at 652 nm of the oxidized product of TMB 38 . It's worth noting the N 2 NB itself has no detectable absorbance at 652 nm (Figures S3), and the redox potentials of N 2 NB-containing water were similar to that of pure water (Table S1). The results showed that the oxidation rates of TMB in water containing ultra-small N 2 NBs were greatly reduced in comparison to that in pure water along with the increase of reaction time, and the absorbance values at plateau were much lower than that in pure water (Fig. 1a), which suggests a strong antioxidant effect of the ultra-small N 2 NBs. In addition, a comparative study indicated that the antioxidant ability of the ultra-small N 2 NBs was equivalent to a common antioxidant, sodium ascorbate, in a concentration between 100 and 200 μM (Fig. 1b).
In our experiments, nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) were employed as complementary means to determine the size distribution and concentration of nanobubbles in water 39 . By monitoring the Brownian Motion of a relatively small number of individual objects, NTA is able to accurately measure the concentration (10 6 -10 9 particles/mL) and size (10-2000 nm) of polydisperse populations 40 . Due to the low light scattering of NBs in water, NTA can test their size distribution in the range of 50-2000 nm, meanwhile determining their concentration. In the case of DLS, the collective diffusion of a larger number of objects is monitored and their average size is calculated. However, DLS only provides a rough size distribution of samples ranging from 0.3 nm to 15 μm without concentration information 41,42 . Figure 1c (upper) showed a typical size distribution of the as-generated N 2 NBs as measured by NTA, with the peaks mostly between 50 and 270 nm. NTA analysis also indicated a NB concentration of 5.42 × 10 7 ± 5.78 × 10 6 particle/ml and an averaged NB size of 152.7 ± 14.1 nm. Figure 1c (bottom) showed two peaks with very strong scatter intensity in the DLS curves, indicating that the sizes mostly centered at 3.62 nm and 255 nm, respectively. The only peak observed in the DLS number percent curve (Fig. 1c, bottom) centered at 3.62 nm, suggesting that these ultra-small NBs made up the overwhelming majority in numbers in the solution.
A degassing experiment was carried out to rule out the possibility that the introduction of impurities during NB generation might have also caused the observed antioxidant effect. By removing most of the N 2 NBs in water after degassing for 24 h under a vacuum of 0.01 atm (Fig. S4), TMB oxidization curves (Fig. 1d) showed that the antioxidation ability of the N 2 NBs water was significantly reduced, clearly confirming that the observed antioxidant effect was originated from the N 2 NBs rather than from impurities. Size dependence of the N 2 NB's antioxidant capability. Since the size of the N 2 NBs generated was widely distributed in the range of 0-300 nm (Fig. 1c), it was plausible to explore if there would have a size dependence for their antioxidant capability. We found that the normal N 2 NBs generated in fresh ultrapure water at room temperature did not inhibit but slightly enhance the oxidation of TMB (Fig. 2a). NTA study showed a typical size distribution of the normal N 2 NBs between 70 and 220 nm (Fig. 2b, upper), a NB concentration of 6.41 × 10 7 ± 1.72 × 10 7 particle/ml, and an averaged NB size of 116.9 ± 14.7 nm. DLS study revealed two strong scattering intensity peaks centered at 142 and 396 nm, respectively (Fig. 2b, bottom). Both NTA and DLS results of normal N 2 NBs showed no detectable NBs with sizes smaller than 50 nm, implying that the antioxidant effect was only caused by the ultra-small NBs (typically < 50 nm). Besides, we found that the ultra-small N 2 NBs transformed from normal N 2 NBs through a freeze-thawing operation also exhibit an antioxidant effect (Fig. S5). In addition to the ultra-small N 2 NBs, ultra-small oxygen (O 2 ) NBs also have a strong antioxidant effect in the TMB oxidation reaction (Fig. S6).
The antioxidation mechanism for the ultra-small NBs. The above results clearly showed that there was a size dependence on the NB's antioxidant capability. Ultra-small N 2 NBs inhibited the oxidization of TMB by hydroxyl radicals, while their clusters or normal N 2 NBs (typically > 50 nm) slightly enhance the oxidation of TMB. The contrasting effects of the small and large NBs on the TMB oxidation seemed difficult to be understood. Currently, our knowledge about the chemical properties of the interfaces of NBs is much poor, it is wise to interpret our observations based on the existing realizations regarding the regulation of oxidation and reduction by gas-water interfaces. Since the electrical surface potential difference of NBs is normally − 20 mV, far smaller than the 3 V at the gas-liquid interface of small water droplets 28 www.nature.com/scientificreports/   www.nature.com/scientificreports/ results from the electrical surface field mechanism proposed by Nam and Richard. Previous studies have shown that, when free radicals and substrates were both enriched at the gas-liquid interfaces, the oxidizing reaction could be accelerated 26,44 . Therefore, we believed that the selective enrichment of ROS at the gas-liquid interface of the NBs might play an important role in our reaction systems. A plausible explanation may be that the surface areas of the ultra-small NBs were so small and had insufficient space for larger substrate molecules to be easily adsorbed, which resulted in the fact that it preferred to enrich more ROS but fewer substrate molecules. The short-lifetime hydroxyl radicals would be enriched at the interface and quenched by themselves (Fig. 3). In contrast, the big surface area of the large NBs (or NB clusters) would enrich both the TMB and the hydroxyl radicals at their gas-liquid interfaces, and enhance the reaction between TMB and hydroxyl radicals as usual. This mechanism also works for another classic hydroxyl radical probe, 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) (Fig. S7). In addition to the hydroxyl radicals, the ultra-small NBs were also found to scavenge superoxide anion radicals (Fig. S8).
Antioxidation of N 2 NBs for hydrophilic substrates. According to our proposed mechanism (Fig. 3), normal NBs enhance oxidation due to that they simultaneously adsorb ROS and hydrophobic TMB at the gasliquid interface, which increases their reaction probability. If this is the case, normal NBs should also exhibit antioxidant effects when substrate molecules that tend to remain in the water phase rather than at the gas-liquid interface are used. To test this hypothesis, dimethyl pyridine N-oxide (DMPO), a commonly-used electron spinresonance (ESR) spin trap, was employed for capturing hydroxyl radicals. DMPO is hydrophilic so that it should present in the water phase. In this experiment, ESR was used to measure the intensity of the oxidized DMPO (DMPO-OH). Results (Fig. 4) showed that DMPO-OH signals in reaction systems containing normal N 2 NBs or ultra-small N 2 NBs were much lower than that of the control group, indicating an antioxidation effect. The results further support our mechanism.

Discussion
To exclude the possible involvement of the reaction system in the antioxidation of the NBs, we employed ultraviolet (UV) radiation [45][46][47] (Figs. 5a and S9) or Fe 2+ instead of Cu 2+ (Fig. S10) to produce hydroxyl radicals from H 2 O 2 . Results also showed a strong antioxidant effect of the ultra-small N 2 NBs in contrast to a slight pro-oxidant effect of the normal N 2 NBs. Thus, we believe that the antioxidant effect of the reductant-free NBs should be mainly ascribed to the ultra-small NBs themself. Compared with conventional reducing agents including reactive nanobubbles 48,49 that are consumptive in the reaction, the use of ultra-small NBs as an antioxidant has certain advantages. First, the ultra-small NBs are stable in pure water 41 and their antioxidant ability could be sustained even under a high-level ROS environment. For example, the ultra-small N 2 NBs kept almost 100% of their antioxidant ability (Fig. 5b) in a system containing high-level hydroxyl radicals that were constantly generated by intense UV radiation of H 2 O 2 in water. In contrast, 1 mM sodium ascorbate was consumed gradually and only kept ~ 0.1% of its original antioxidant ability (Fig. 5b) under the same condition. Second, according to the proposed antioxidation mechanism, there would be no harmful oxidized products remaining after quenching ROS with the ultra-small NBs. We believe

Conclusion
In summary, an antioxidant effect of ultra-small NBs has been explored. Our results indicated that the ultra-small NBs had an obvious effect to inhibit the oxidation of hydrophobic substrates (TMB) or hydrophilic substrates (DMPO) caused by hydroxyl radicals. Since there was no special chemical reducing agents added in the reaction system, the antioxidation ability of the ultra-small NBs could be used safely in living systems and might find its potential applications in relieving oxidative stress in organisms including human beings. In addition, our results may also provide a new scientific view to the controversial issue about the claimed healthy effects of some natural or 'functional' water 51 , since NBs are believed to exist ubiquitously in nature. Further explorations should be conducted in developing techniques to prepare ultra-small NBs with higher concentrations and more precise regulation of the size distributions to fit the antioxidation demands in many practical applications.  www.nature.com/scientificreports/

Materials and methods
Materials. Ultrapure water was prepared from an ELGA LabWater (ELGA Classic-PURELAB) instrument.
Generation of NBs. The NBs were generated by a compression-decompression method in ultrapure water that was previously reported 37 . The experiment was carried out in a custom-made metal chamber with pressure control. First, ultrapure water was placed into the chamber, and gas (N 2 or O 2 ) was introduced into the chamber to a pressure of 0.6 MPa. Then, the pressure in the chamber was slowly reduced (20 sccm) to normal pressure (1 atm). NBs were generated in ultrapure water that was either at room temperature or at 0 °C that was prepared from a mixture of ice and water.
Analysis of NBs. The nanoparticle tracking analysis (NTA) system (NS300, Malvern, UK) was used to analyze the number density and size of the prepared NBs in water. NTA 3.4 software was used to capture and analyze data. Besides, a dynamic light scattering (DLS, nano-ZS90, Malvern) instrument was also employed to detect the scattering light intensity and number (%) of NBs.  (1 × 1 × 5 cm 3 ) was placed 30 cm away from a UV lamp (20 W) and was irradiated at a wavelength of 256 nm for 10 min. The solution's absorbance value at a wavelength of 652 nm was measured at the end of the radiation.

Data availability
All data generated or analysed during this study are included in this published article and its supplementary information files.